Since the elucidation of the viral-encoded factors required for lytic DNA replication it is clear that UL84 has emerged as the central factor proposed to be involved in the regulation and coordination of initiation of viral DNA synthesis. Our laboratory and others demonstrated that UL84 is a multifunctional phosphorylated and ubiquinated protein that displays nucleocytoplasmic shuttling, interacts with viral proteins IE2, UL44, UL83 and binds to RNA and DNA. Recent proteomics data from our laboratory revealed the cellular and viral binding partners for UL84 in infected cells. These discoveries have now illuminated new areas of research with respect to understanding the precise role of UL84 in HCMV growth. We have now demonstrated that UL84 interacts with the cellular kinase Casein Kinase II (CK2) and two amino acid residues within the UL84 mediated this interaction. Mutation of these two amino acid residues (aa 148 and 157) results in the inability of CK2 to interact with UL84. Mutation of these residues renders UL84 incapable of complementing oriLyt-depended DNA replication, suggesting that phosphorylation is an essential modification for the replication function of UL84. Additionally, we also show that UL84 interacts with UL44, the pol accessory protein. The binding of UL84 with UL44 suggests that the pol accessory protein may act as a cofactor for the interaction of UL84 with oriLyt and possibly recruits UL44 to oriLyt as part of the replication complex. Additionally, we now show that UL84 interacts with CCAAT/enhancer binding (C/EBP) transcription factor-binding sites within oriLyt in the absence of C/EBP, and these sites are essential for oriLyt-dependent DNA replication. This is the first reporting of a defined binding site for UL84 in oriLyt. We also demonstrate that the nuclear export activity of UL84 is essential for oriLyt-dependent DNA replication and in the context of the virus genome. This suggests that UL84 interacts with viral and/or cellular mRNA and this interaction plays an essential role in virus replication. Using an UL84-RNA pull down assay we have identified the viral mRNA encoding IRS1 as one transcript associated with UL84. This proposal will seek to determine a role for the posttranslational modifications of ubiquitination and CK2-mediated phosphorylation and elucidate the complete set of cellular/viral factors interacting with specific elements within oriLyt. Lastly, we will determine specific RNA species interacting with UL84 and how nucleocytoplasmic shuttling contributes to the regulation of DNA synthesis. PUBLIC HEALTH RELEVANCE: Human cytomegalovirus (HCMV) is a beta herpesvirus that is normally asymptomatic in immune- competent hosts. HCMV UL84 is a central factor apparently controlling initiation and regulation of DNA synthesis. This proposal will characterize posttranslational modifications of this protein and evaluate viral/cellular factors interacting with oriLyt. Additionally we will explore the interaction of UL84 with other viral encoded factors and identify the role of nucleocytoplasmic shuttling in the activity of UL84.